Cardiovascular disease (CVD) is the leading cause of human deaths worldwide. It is estimated that, by 2015, approximately 20 million people will die from cardiovascular disease (CVD). Numerous cardiovascular diseases (CVDs) (such as myocardial infarction and apoplexy) are the leading complications of atherosclerosis (AS). So far, the pathogenesis of atherosclerosis has not been fully understood. Abnormal lipid metabolism is one of the main risk factors that cause this disease. High-level of low density lipoprotein (LDL) and low-level of high density lipoproteins (HDL) are the two most important risk factors. Traditional strategies of treatment are to reduce the content of total cholesterol (TC) and low density lipoproteins cholesterol (LDL-C) in blood plasma. Statins are the preferred lipid-lowering drug at present. However, they cannot eliminate existing plaques deposited on artery wall, or fundamentally cure atherosclerosis (AS). More and more scientists in different countries have turn to the other risk factor, low-level of high density lipoprotein (HDL). Epidemiological studies indicate that, the level of high density lipoproteins (HDL) in blood plasma is in negative correlation to the incidence of coronary diseases. It is believed that high density lipoprotein contributes to the prevention of atherosclerosis. Treating atherosclerosis through improving the level of high density lipoprotein is a new approach for treating acute coronal atherosclerosis diseases that is emerging in pharmaceutical industry. It is called high density lipoprotein targeted therapy. Apolipoprotein A-I (apo A-I) is the main proteic component of high density lipoproteins. Apolipoprotein A-I is synthesized in liver and small intestine. The primary translation product is the preproprotein (preproapo A-I) containing 267 amino acid residues. Preproprotein is then processed into the proprotein (proapo A-I), through the cleavage of an octadeca peptide by signal peptidase. The proapo is secreted and processed into mature plasma apolipoprotein A-I through cleavage of a hexapeptide (Arg-His-Phe-Trp-Gln-Gln) by specific extracellular converting enzymes. The mainly mechanism of action of apolipoprotein A-I is to promote the cholesterol efflux, antioxidation, and to decrease platelet aggregation.
Apolipoprotein (apolipoprotein A-IM, apoA-IM) is a natural mutant of apolipoprotein A-I (Arg173-Cys). Compared with apolipoprotein A-I, the loss of Arg173 leads to the reduction of content of α-helix and the enhancement of the capability to bind lipid. Apolipoprotein A-I-Milano tends to form a dimer (A-IM/A-IM). This dimer stimulates the reverse transport of cholesterol, and thus the clearance of cholesterol, more efficiently than apolipoprotein A-I. Compared with apolipoprotein A-I, apolipoprotein A-I-Milano more efficiently decrease the oxidation of low density lipoprotein. At present, apolipoprotein A-I-Milano is the only pharmaceutical protein that is shown to remove the thrombus deposited on artery wall, with broad application prospect.
It is reported in the Journal of the American Medical Association (JAMA) recently that, apolipoprotein A-I-Milano effects changes of artery atherosclerosis lesion with unprecedentedly speed and amplitude and little side effect. With various application prospects, it has become the focus of pharmaceutical research and industrial competition worldwide. Pfizer, the largest pharmaceutical company in the world, estimates that any drug reversing artery plaque may be a billion dollar business. Therefore, the development of apolipoprotein A-I-Milano will definitely bring about enormous economical and social benefits, as well as enhance the competitive strength in the field of drug development against cardiovascular diseases and atherosclerosis diseases.
In addition, data obtained from small-scale clinical trials reveal that the clinical dosage of apolipoprotein A-I is 5-6 g per treatment course. The high therapeutic dosage of apolipoprotein A-I and the high prevalence of atherosclerosis suggest huge market demand and also an opportunity for the development of apolipoprotein A-I-Milano. At present, apolipoprotein A-I-Milano is produced by Eperion, US by means of biosynthesis, which is of high cost and low yield and undesirable for large-scale production. The recombinant expression of the protein in bacterial system is generally attractive. However, the yield is low, and Escherchia coli endotoxin tends to form tight complex with apolipoprotein A-I-Milano. Besides, the protein purification method is expensive and poor in safety. Therefore, there is the need for a method of producing apolipoprotein A-I-Milano with high yield and efficiency.
Plant bioreactor, also called molecular medicine farming, refers to the large-scale production of heterologous proteins of importance and commercial value, especially medical proteins used for the treatment or diagnosis of diseases, by a plant biological system. Mammalian antibodies were successfully expressed in transgenic plants for the first time in 1989. Both the heavy and light chains were expressed and correctly assembled in transgenic tobacco, demonstrating for the first time the possibility to use plant as a bioreactor. Since then, researches directed to transgenic plants have been rising. Many other medical proteins have been expressed in different plants sooner or later, such as hirudin, interferon, human albumin, and functional antibodies. Plants already used in plant bioreactor research include tobacco, Arabidopsis thaliana, soybean, wheat, rice, rape, potato and tomato, etc.
SemBioSys Genetic, Inc, a Canadian biotechnology company developing protein drug combinations for metabolic and cardiovascular diseases, filed a patent application in China (CN1906296A) regarding the method for producing apolipoprotein A-I and apolipoprotein A-I-Milano with transgenic Carthamus tinctorius and Arabidopsis thaliana, in which a chimeric nucleic acid construct is introduced into Arabidopsis thaliana or Carthamus tinctorius. Apolipoprotein A-I and apolipoprotein A-I-Milano is expressed in seeds upon seed setting. Arabidopsis thaliana is an annual or biennial herb. It has the smallest genome among all plants. Due to its high generic homozygosity, high mutation rates may be achieved upon physical or chemical treatments, providing various metabolic deficiency phenotypes. Thus Arabidopsis thaliana represents a good material for genetics research, and is called “the fruit fly of the plant world”. Though Arabidopsis thaliana is widely used in experimental contexts, it is not utilized in large-scale production. Carthamus tinctorius is an annual herb. Its seed can be used for oil extraction, and thus it is an important oil crop. It is distributed in the temperate zone. In China, it is mainly distributed in the Northwest (in particular Xinjiang and Tibet), and then North China and Northeast regions. Carthamus tinctorius suffers from the disadvantage of relatively low yield per mu (120-150 kg) and suboptimal oil content of the achene (34˜55%), resulting in low productivity of the end product protein and a high cost.
Therefore, there is still the need for a method for producing apolipoprotein A-I and apolipoprotein A-I-Milano with stable and high yield, low cost, and simple procedures.